Methods for separating nucleic acids with graphene coated magnetic beads

ABSTRACT

Methods for separating and identifying nucleic acids utilize carbon coated magnetic beads. The method teaches that multivalent cations promote binding of single stranded nucleic acids to the beads and that the single stranded nucleic acids can be released with the addition of chelating agents that bind the multivalent cations such as EDTA. The method further teaches that fragile single stranded nucleic acids, such as RNA, can be stored on the surface of the beads. Lastly, the method also teaches that by iteratively adding complimentary DNA oligos, single stranded nucleic acids can be quantified or individually isolated using the carbon coated magnetic beads.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/434,470, filed Feb. 16, 2017, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 62/295,985, each of whichis incorporated herein in its entirety by reference.

FIELD OF THE DISCLOSURE

This disclosure relates to nucleic acid purification and isolation,magnetic bead biological separations, and carbon and its allotropes

BACKGROUND

The need for effective techniques for isolating single stranded nucleicacid components from associated biological materials persists in avariety of research and development endeavors. Heretofore, nucleic acidseparation techniques can include various chelating agents, solidsupport columns using fibrous or silica matrices to bind nucleic acidsand/or magnetic beads to which nucleic acid can bind. The isolationmethods presently in use do not provide effective yield in allinstances. Thus, it would be desirable to provide a method and materialthat can provide more effective and efficient nucleic acid isolation.

SUMMARY

An improved method for separating single stranded nucleic acids from asample that includes the steps of: providing a mixture containingnucleic acids; and providing carbon coated material; providing achaotropic salt such as guanidinium thiocyanate; providing alcohols suchas ethanol or organic solvents; and creating a complex of the carboncoated material and nucleic acids. In certain embodiments, thecarbon-coated material employed can be in the form of magnetic beads,silica gel or various polymeric materials. The method disclosed hereincan also include the steps of removing the carbon coated materialcomplexed with nucleic acids from the resulting mixture by means ofmagnetic separation or centrifugation. The method may further includethe steps of washing the carbon-coated material and nucleic acidscomplex with alcohol based solutions. The method may also include thestep of reversing the complexation of the nucleic acids with thecarbon-coated material by the addition of water or aqueous liquids.

In certain embodiments, carbon coated magnetic beads may be employedthat can have a magnetic core coated surrounded by a suitable carbonmaterial. In other embodiments, the carbon coated material may have anon-magnetic core such as silica coated with a suitable carbon material.

BRIEF DESCRIPTION OF THE DRAWINGS

The various features, advantages and other uses of the present apparatuswill become more apparent by referring to the following detaileddrawings.

FIG. 1 is a schematic drawing for how single stranded nucleic acids areisolated.

FIG. 2 is a flow chart of an embodiment of the process disclosed hereinand depicted in FIG. 1.

FIG. 3 is a schematic drawing for how a single stranded nucleic acid ina sample may be isolated and may also be quantified.

FIG. 4 is a flow chart for the process of how a single stranded nucleicacid in a sample may be isolated and may also be quantified.

FIG. 5 is a transmission electron micrographs of a carbon coatedmagnetic beads which may be used in this process.

FIG. 6 is a flow chart of a process for separating and producing asingle stranded nucleic acid.

FIG. 7 is a gel electrophoresis image showing nucleic acids extractedfrom MCF7 immortalized breast cancer cells using 125 μl of: 2M, 3M, 4M,5M, and 6M guanidinium thiocyanate with 500 μl of 70% ethanol and 30% pH8 Tris.

FIG. 8 is a gel electrophoresis image showing nucleic acids extractedfrom MCF7 immortalized breast cancer cells using 125 μl of 6Mguanidinium thiocyanate without ethanol added and with 500 μl of 70%ethanol and 30% pH 8 Tris.

FIG. 9 is a gel electrophoresis image comparing nucleic acids extractedfrom MCF7 immortalized breast cancer cells using a buffer composed of125 μl of 2M guanidinium thiocyanate and 500 μl of 70% ethanol andcarbon coated magnetic beads compared with a nucleic acid isolation kitsold by Fisher Scientific.

FIG. 10 is a flow chart for the process of how sequence specificanalysis may be performed using chaotropic salts, alcohols, and carboncoated materials.

DETAILED DESCRIPTION

An improved method of separating single stranded nucleic acids from amixture is disclosed herein. The method includes the steps of providinga sample containing single stranded nucleic acids and contacting thesample containing single-stranded nucleic acid with a carbon-coatedmaterial, at least one chaotropic salt, and at least one alcohol toeffect separation.

An embodiment of the separation process as disclosed herein isillustrated in FIG. 1 and described as a flow chart in FIG. 2.

As depicted FIG. 1, the process 100 begins with a collected sample 12containing nucleic acids and a suitable solvent, such as would be foundin in various microorganisms, plant and animal specimens such asmammalian tissue, a non-limiting example of which is human liver tissue.The collected sample 12 can be maintained in a suitable vessel such asthat depicted in FIG. 1 at reference numeral 15. The process asdisclosed herein can take placed in a suitable reaction vessel orcontainer or semi-permeable matrix. In the embodiment depicted in FIG.1, the reaction vessel is an elongated tube.

To this sample 12 is added a chaotropic material 14 such as a chaotropicsalt solution and carbon-coated materials 16. This creates a mixture ofnucleic acids, chaotropic materials 14, and carbon-coated materials 16as at reference numeral 17. Without being bound to any theory, it isbelieved that contact between the nucleic acids present in the sampleand the chaotropic material results in separation of single strandednucleic acid material 18 a and double stranded nucleic acid material 18b into the solution with single and double stranded nucleic acidmaterial collectively referend to as reference numeral 18.

To this resulting mixture is added an alcohol as at reference numeral19. The alcohol of choice can be a short-chain alcohol such as ethanolor the like. Once the nucleic acid material 18, chaotropic material 14,carbon-coated material 16, and solvent are present together, thesingle-stranded nucleic acids 18 a present in the sample complex withthe carbon-coated material 16 and create a nucleic acids-carbon complex16A as at reference numeral 19.

While the embodiment depicted in FIG. 1, depicts a process in which thevarious material components are introduced in a specific sequentialorder, it is considered within the purview of this disclosure that thefour materials may be provided in any order such that the four materialsare present together before the separation step such as that denoted atreference numeral 21. Where desired or required, the four components canbe admixed to ensure contact between the various components.

Once all four material components are in admixture together, the admixedmaterials can remain in contact with one another for a suitable periodsufficient for the nucleic acids present in the initial sample tocomplex with the carbon coated material. Where desired or required, thecontact interval can be between 1 second and 5 minutes. In certainembodiments, the complexing period can be between 10 seconds and 5minutes.

The carbon-coated material can be configured in any suitable size andshape that would facilitate complexing with the desired nucleic acids.Non-limiting examples of such configurations include beads in which eachindividual bead has an interior core and an outer surface, the beadscomposed of one of the following: a magnetic metal containing at between20 and 40% by weight of a metal selected from the group consisting ofNi, Fe, Co, or mixtures thereof, silica or polymeric substrates, andwherein the carbon coating is present as a carbon layer on at least aportion of the outer surface of the bead, the carbon layer comprising atleast one of graphene, pyrolytic carbon or a mixture of graphene andpyrolytic carbon, and wherein the multivalent cations are alkali earthmetals or alkali earth metal salts or mixtures thereof. Thus, in certainembodiments, it is contemplated that magnetic particles can be used asthe carbon-coated material 16. In situations where carbon coatedmaterials have a magnetic substrate, it is contemplated that exposure toa suitable magnetic field such as that produced by magnet 20 willproduce separation between the nucleic acid carbon material complexes16A and the liquid components present resulting in the development of asupernatant solution 26 and collected nucleic acids complexed withcarbon material 16A. This may be accomplished by placing a magnet 20near a specific location on the exterior of the reaction vessel orcontainer as at reference numeral 22 to collect the carbon coatedmaterial and facilitate the process of separation as at referencenumeral 28 and illustrated in FIG. 1. This results in a supernatantsolution 26 that comprises double stranded nucleic acid, as well asother materials that may be present in the initial sample as depicted inreference numeral 23A. The complexes 16A can be transferred to aseparate vessel for additional processing as at reference numeral 23B.

Where desired or required, the complexed carbon material 16A can bewashed one or more times to ensure that all the supernatant liquidcontaining the cellular contaminants is removed. The washing step may beperformed by contacting the separated material with one or more portionsof a solution containing one or more of alcohols. The washing step maybe performed multiple times to ensure that all the cellular contaminantspresent in the original supernatant have been removed. After severalwashes with a material such as an ethanol solution, the wash step can becompleted by removing all of the wash solution. Air drying or otherdrying means may be employed to speed removal of the alcohol solutionfrom the complex of the carbon coated material and nucleic acids. Purewater or water with less than 1M salt can then be added at referencenumeral 30 in order to facilitate removal of the nucleic acids fromcomplex with the carbon-containing material. In certain embodiments, itis contemplated that 50 μl or 100 μl of pure water can be employed insituations in which around 1 to 5 million cells are used as the sourcesample, yielding a solution with about 10 to 100 μg of single strandednucleic acid (RNA) dissolved in the pure water solution.

An embodiment of the separation process is illustrated in the flow chartin FIG. 2 in which nucleic acids are separated from a mixture using achaotropic material such as a chaotropic salt solution, a polar solventsuch as ethanol, and a carbon coated material.

As depicted in FIG. 2 at reference numeral 200, a sample containingnucleic acids is contacted with a chaotropic material such as achaotropic salt to form an aqueous solution as at reference numeral 210.Carbon-coated materials are introduced to the aqueous solution atreference numeral 212. Alcohols are introduced to the aqueous solutionat reference numeral 214. It is also considered to be with in thepurview of this disclosure that the four materials may be provided inany order such that the four materials are present together prior to anyseparation operations on the resulting solution. The process asdisclosed herein can occur in a suitable reaction vessel or container.

Once all four material components are in mixture together, the materialscan remain in contact with one another for a suitable period sufficientfor the nucleic acids present in the initial sample to complex with thecarbon coated material such as carbon beads. Where desired or required,the contact interval can be between 1 s and 5 min.

Once formed, the complexes of nucleic acids and carbon-containingmaterial are separated from the supernatant liquid as at referencenumeral 616. The resulting supernatant liquid can contain the initialliquid components in addition to extraneous cellular component material.

In certain embodiments, it is contemplated that magnetic beads coatedwith carbon can be used as the carbon-coated material. Separation can beaccomplished by one or more mechanisms including, but not limited to,decanting, filtration and various mechanical separation methods. Insituations where carbon coated beads had a magnetic substrate, it iscontemplated that the separation step utilizing magnetic beads can beaccomplished by placing a magnet near the exterior of reaction vessel orcontainer to collect the carbon coated material and facilitate removalof the remaining solution. If the substrate onto which the carbon iscoated is not magnetic, physical removal, centrifugation, or othermethods may be used to separate the carbon material.

Where desired or required, the complexed carbon material can be washedone or more times to ensure that all the supernatant liquid containingthe cellular contaminants is removed as at reference numeral 218. Thewashing step may be performed by contacting the separated material withone or more portions of a solution containing one or more of alcoholsenumerated previously. The washing step may be performed multiple timesto ensure that all the cellular contaminants present in the originalsupernatant have been removed. After several washes with a material suchas an ethanol solution, the wash step can be completed by removing allof the wash solution. Air drying or other drying means may be employedto speed removal of the alcohol solution from the complex of the carboncoated material and nucleic acids. Purified water or water with lessthan 1M salt can then be added in order to facilitate removal of thenucleic acids from complex with the carbon-containing material atreference numeral 220. In certain embodiments, it is contemplated that,50 μl or 100 μl of this water can be employed to release the singlestranded nucleic acids.

The method as disclosed herein can also be employed to isolate andquantify a single stranded nucleic acid material with specificsequencing. A non-limiting exemplary method is illustrated in FIG. 3. Abiological material to be analyzed is collected as at reference numeral312, which may be the result of process 100. The sample in question cancontain target single stranded nucleic acids 314 as well as doublestranded nucleic acids 315 some of which are nucleic acids that containa desired sequence for quantification together in a suitable solution319.

Specific probes 316 can be added to the sample as illustrated atreference numeral 318 and allowed to react with any complementarynucleic acid material that is present in the sample 314 to form a duplexcomprising the probe and the target. At step 317, a chaotropic salt, asuitable carbon-coated material 320, and an organic solvent are added tothe solution if not already present. Once all four material componentsare in admixture together, the materials can remain in contact with oneanother for a suitable period sufficient for the single stranded nucleicacids 314 present in the initial sample to complex with the carboncoated material 320. Where desired or required, the contact interval canbe between 1 second and 10 minutes, with intervals between 10 secondsand 5 minutes being employed in certain embodiment. The duplexes whichcontain both a probe 316 and a double stranded nucleic acid 315 targetremain in solution 319.

In situations where carbon coated beads 320 have a magnetic substrate,it is contemplated that separation of the supernatant containing theduplex double stranded nucleic acids 315 and single stranded nucleicacids 314 complexed with carbon material 320 may be accomplished byplacing a magnet 322 near the exterior of reaction vessel or container324 to collect the carbon coated material as a pellet and facilitate theprocess of separation.

It is contemplated that the complementary nucleic acid material may bemodified with a protein which may be used to isolate strands withspecific sequencing. Once separated the duplex double stranded nucleicacids 315 modified with complementary proteins to those on thecomplementary nucleic acids may be added to the mixture to concentrateor isolate the target sequences. By way of non-limiting example, biotinmay be attached to the complementary nucleic acids and streptavidin maybe attached to a substrate or magnetic beads.

Separation of the supernatant liquid from contact with the carbon coatedbeads 320 yields a solution of the duplex material 316 that is amenableto various analytic techniques including, but not limited to florescenttag detection 336, simple PCR 338, magnetic sensor detection 340 and thelike.

It is further conceived that the method as disclosed herein can beemployed to perform sequence specific analysis and one non-limitingexample of such a procedure is further illustrated in the flow chartdiagram of FIG. 10 as at reference numeral 1000. A suitable sample ofsingle stranded nucleic acids can be collected as at reference numeral1010. It is contemplated that the single stranded nucleic acid can bederived from any suitable source or by any suitable method; one suitableprocess for separating and producing single stranded nucleic acidprocess disclosed herein in FIG. 6.

In the process as disclosed, at least one complimentary nucleic acidprobe can be added to the collected material as at reference numeral1012. It is contemplated that the nucleic acid probe can be a suitablediagnostic material. Non-limiting examples of such material include DNA,RNA, as well as synthetic nucleic acids such as peptide nucleic acids.If desired or required, the added material can be allowed to react withthe single strand nucleic acid for a suitable interval.

Once any optional reaction interval has elapsed, alcohols, chaotropicmaterials such as chaotropic salts, and a carbon coated material such asthose described herein can be added to the sample to initiate binding ofthe single stranded nucleic acids to the carbon-coated material as atreference numeral 1014. In certain embodiments, it is contemplated thatthis step will resemble the process step outlined in with regard to FIG.6. It is also considered with in the purview of this disclosure thatwhere little or no protein or cellular contamination is present, theaddition of chaotropic materials such as chaotropic salts may be reducedor eliminated entirely, It is contemplated that the complex of thecarbon coated material and single stranded nucleic acids that isproduced may also contains complimentary probes which were added atreference numeral 1012 but do not find a target with which to hybridize.

Where desired or required, the resulting material can be permitted toform suitable complexes of the single stranded nucleic acid to whichcomplementary probes have bonded and carbon-containing material as atreference numeral 1016. The formation interval can be any suitableperiod between 10 seconds and 24 hours in certain embodiments.

The resulting complex of nucleic acid and carbon coated material canthen be separated as at reference number 1018. Separation can beaccomplished by one or more mechanisms including, but not limited to,decanting, filtration and various mechanical separation methods. Insituations where carbon coated beads have a magnetic substrate, it iscontemplated that the separation step utilizing magnetic beads can beaccomplished by placing a magnet near the exterior of reaction vessel orcontainer or semi-permeable matrix to collect the carbon coated materialand facilitate removal of the remaining solution.

The resulting supernatant can be removed for further analysis as desiredor required as at reference numeral 1020 contains double strandedhybrids of the target and complimentary nucleic acids, which may bequantified as at reference numeral 1022. Non-limiting examples ofsuitable quantification methods include by PCR amplification referencenumeral 1024, or by labeling the synthetic nucleic acid with adetectable fluorescent or magnetic tag as at reference numeral 1026. Itis within the purview of this disclosure that the synthetic nucleicacids may be labeled before being added to the sample as at referencenumeral 1012. It is also contemplated that the probes can be labeledafter the supernatant is separated from the nucleic acid-carbon complexas at reference numeral 1026.

The nucleic acids that are complexed with the carbon material may bewashed and eluted from the carbon coated material as at referencenumeral 1028. Where desired or required, the complexed carbon materialcan be washed one or more times to ensure that all the supernatantliquid containing the cellular contaminants is removed as at referencenumeral. The washing step may be performed by contacting the separatedmaterial with one or more portions of a solution containing one or moreof alcohols enumerated previously. The washing step may be performedmultiple times to ensure that all the cellular contaminants present inthe original supernatant have been removed. After several washes with amaterial such as an ethanol solution, the wash step can be completed iscompleted by removing all of the wash solution. Air drying or otherdrying means may be employed to speed removal of the alcohol solutionfrom the complex of the carbon coated material and nucleic acids. Purewater or water with less than 1M salt can then be added in order tofacilitate removal of the nucleic acids from complex with thecarbon-containing material. In certain embodiments, it is contemplatedthat, in certain embodiments, 50 μl or 100 μl of pure water can beemployed in situations in which around 1 million cells are used as thesource sample. The resulting sample of single stranded nucleic acidseluted as at reference numeral 1028 is suitable for use as a sample at1010 and the process may be repeated.

The carbon-coated material can be composed of a suitable substratehaving an outer surface with a suitable carbon material covering atleast a portion of the outer surface of the substrate. The resultingcarbon-nucleic acid complex may be separated from the remainingmaterials by various separation techniques. The techniques employed canbe chosen based on factors such as the nature of the of the substratematerial employed.

As used herein the term “nucleic acid probe” or “antibody probe” refersto a chain of nucleic acids selected so that it contains bases that arecomplementary or have affinity with corresponding positions in at leastone section of the length of the nucleic acid chain under study. Thecomplimentary nucleic acid probe may be longer than the target and haveadditional bases with an arbitrary sequence. Anti-nucleic acid antibodymay have additional chemical modifications such as biotin tag. Thecomplimentary probe may be synthetic nucleic acids such as peptidenucleic acid (PNA) or Morpholino and locked nucleic acid (LNA) or glycolnucleic acid (GNA) or threose nucleic acid (TNA).

Also disclosed is a composition that comprises a carbon coated substrateand single-stranded nucleic acid complexed therewith.

The carbon coated material as disclosed herein is one that will complexwith single stranded nucleic acid material present in a sample. Thesingle stranded nucleic acid material can include various naturallyderived and/or synthetically prepared nucleic acids such as RNA,including any added complimentary nucleic acid probes if the target forthese probes is not present in the sample under study. Particles withthe single-stranded nucleic acids bound to the magnetic beads may beremoved by application of a magnetic field, centrifugation,precipitation and the like.

In certain embodiments, the carbon coated material will include asubstrate that is configured as a particulate material such as beads, orother spheroid bodies. When the substrate is configured as beads orother particulate geometries that are nanoparticulate in size; having adiameter between 20 and 200 nm or between 10 nm and 100,000 nm incertain embodiments.

In certain embodiments, the carbon material can include one of more ofthe following: pyrolytic carbon, graphite, graphene. In certainembodiments, the suitable material is a material with a metal core thatis surrounded by a carbon coating in which the carbon creates agenerally seamless conformal coating. A suitable carbon coated materialthat can be used in the process as disclosed herein can be one such asthe bead configuration presented in FIG. 5. Referring to FIG. 5, thereis depicted a transmission electron micrograph in which a metal corecomposed of Fe₆₅Co₃₅ is surrounded by a carbon coating. This carboncoating has multiple layers, in which each layer is similar to grapheneand there may be coupling between the layers. The presented carboncoated material structure is particularly well suited to the describedprocess because the carbon creates a conformal coating without a seamaround the metal core. Exposure of the metal core to water hasundesirable effects due to oxidation of the metal. Oxidation of themetal may lead to undesirable properties such as binding DNA andreduction of magnetic moment. Suitable magnetic material that can beemployed as the substrate is available commercially from MetastableMaterials, Inc., doing business as Life Magnetics.

Carbon coated magnetic materials may be prepared by the lasermanufacturing process disclosed in WO2015095398A1 which is disclosedherein by reference. It is contemplated that carbon coated materials canbe prepared by positioning a laser is incident on target in a solventsuch that the pulsed laser produces laser pulses having a pulse durationgreater than 1 ps at a wavelength between 200 nm and 1500 nm at a pulserepetition rate of at least 10 Hz and a fluence greater than 10 J/cm².The laser beam may be scanned across the surface of the target; i.e.,the desired core material (Silica, magnetic metal and the like).Non-limiting examples of satiable magnetic metal material includesmetals and alloys containing iron, nickel, cobalt, or the like as wellas mixtures and alloys of the aforementioned. The solvent employed canbe an organic solvent which contains carbon and in certain embodimentswill be composed of xylenes, toluene, benzene, or mixtures thereof.Where desired or required, the organic solvent may further include anyhydrocarbon containing benzene rings. In certain embodiments, themagnetic beads employed will have a saturation magnetization between 10and 80 A m²/kg (emu/g); a coercive force between 0.8 and 15.9 KA/m, anda sedimentation rate between 2% and 15% in 30 minutes.

It is envisioned that the chaotropic material imparted may be may beprovided independently in certain embodiments or may be introduced incombination with various other components depending on the nature of thesample containing the single-stranded nucleic acids. In certainembodiments, the chaotropic material composition may further containcomponents on or more additional components. These include, but are notlimited to one or more of the following: ethylenediaminetetraacetic acid(EDTA), dithiothreitol (DTT), 2-mercaptoethanol (BME), Lithium Chloride(LiCl), Urea, N-lauroylsarcosine, IGEPAL 630, NP-40, SDS (sodium dodecylsulfate), TCEP (tris(2-carboxyethyl) phosphine), Tributylphosphine (inN-methyl-2-pyrrolidinone), glycogen or the like. Where desired orrequired this compound can be present t a concentration of between0.001% and 1% by volume. Without being bound to any theory, it isbelieved that the compound functions to inhibit protein activity. Thechaotropic composition can also include a suitable buffer compoundpresent at a concentration suitable to impart buffering characteristicsto the composition.

Non-limiting examples of suitable buffer compounds includetris(hydroxymethyl)aminomethane (Tris), citric acid, sodium citrate,(3-(N-morpholino) propanesulfonic acid) (MOPS),2-(N-morpholino)ethanesufonic acid (MES) as well as mixtures of thesecompounds. In certain embodiments, the buffer compound(s) may be presentat a concentration between 50 mM and 500 mM to buffer the pH of thesolution. The composition can also include detergents such as TritonX-100, or sodium dodecyl sulfate (SDS) or Tween-20 or Tween-40 orN-lauroylsarcosine or IGEPAL 630 or NP-40 where desired or required.

In one non-limiting example of a suitable chaotropic material that canbe employed one or more embodiments of the method as disclosed hereinmay be a solution of chaotropic salts which also contains between 1 to100 mM EDTA, 1 to 100 mM of a buffer composed of citric acid, sodiumcitrate, and/or Tris, 0.01 to 5 volume % Triton X-100, and 0.1 to 5 mMDTT. The pH of the solution may be between 3 to 10. In one exemplarycase, the chaotropic material is a chaotropic salt solution of 4Mguanidinium thiocyanate, 20 mM citric acid buffer, and the pH isadjusted to 5.

It is further envisioned that, in certain embodiments, additional washsteps may be necessary as would be the case for biological substanceshaving a high protein content such as blood. The process as disclosedherein may include one or more additional washing steps in which thewash solution employed may contain suitable materials employedindividually or in combination such as reducing agents, ethanol, andchaotropic salts such as guanidinium thiocyanate, to aid in thesolubilization and removal of the protein. For example, an additionalwash step may be included which uses a wash solution composed of 0.6M to2M chaotropic salts such as guanidinium thiocyanate, and 30% to 60%alcohol by volume. If this additional wash step is included, it iscontemplated that a final washing step with a solution of ethanolwithout chaotropic salts will still occur.

It is envisioned that the chaotropic material may be chaotropic saltssolutions other than guanidinium thiocyanate, such as guanidiniumchloride, guanidinium bromide, guanidinium fluoride, guanidine acetate,guanidine sulfate, guanidine nitrate, guanidine carbonate, or may beother chaotropic compounds such as guanidine hydrochloride, urea,thiourea, lithium acetate, or lithium perchlorate and these compoundsmay have different efficacy in the process, it is expected differentconcentration ranges may be required depending on the efficacy of thesalt. The chaotropic material guanidinium thiocyanate is used as a saltsolution such that the concentration of the guanidinium thiocyanate saltis 0.1 to 1M when the four materials are present together at referencenumeral 21. These other compounds may be expected to have similareffects but at different concentrations, meaning that the concentrationof chaotropic salts may be 0.01M to 4M at reference numeral 21 dependingon the identity of the chaotropic salt, which other components arepresent, and the pH of the solution.

In the process as disclosed, a short chain alcohol such as ethanol canbe added to the composition in an amount sufficient to facilitatecomplexation of single stranded nucleic acids with carbon materials suchas carbon-coated magnetic beads. In certain embodiments, the short-chainalcohol can be added such that the final concentration of the chaotropicmaterial such as guanidinium thiocyanate is between 0.1M and 1M and theshort-chain alcohol composes between 40 vol % and 80 vol % of theresulting solution employed at reference numeral 21 or reference numeral615 in the flow chart. It has been found that under these conditions,single stranded nucleic acids complex with the carbon coated magneticbeads selectively rather than complexing with double stranded nucleicacids. It is also conceived that alcohols other than ethanol may beused, and depending on the alcohol, the ideal percentage of the totalvolume may be different. Alcohols such as propanol, methanol, butanol,phenol, and the like may replace ethanol. It is further conceived that arange of 20 vol % to 90 vol % alcohols may be used.

In certain embodiments, it is contemplated that the mixture whichcontains the sample containing nucleic acids, the carbon-coatedmaterial, the chaotropic material, and the alcohols, has carbon-coatedbeads at a concentration between 1×10⁵ nanoparticulate beads per ml and1×10²⁰ nanoparticulate beads per ml.

It is contemplated that the process and material as disclosed herein canbe employed is to store RNA long term. RNA is known to be fragile and todegrade upon exposure to various enzymes and proteins extant in theenvironment. One non-limiting example of such material is ribonuclease(RNase). It has been found that single-strand nucleic acids such as RNAbind to the surface of the carbon-coated magnetic beads as disclosedherein in a manner that renders them resistant to interaction withenzymes such as RNase. Thus, the bio-complex of a single-strand nucleicacid such as RNA and the carbon coated magnetic beads as disclosedherein can provide a stable long-term storage vector for fragilesingle-strand nucleic acids.

It is contemplated that the stable stored single-strand nucleic acidmaterial can comprise 10-5000 ng of nucleic acids per microliter and50-500,000 ng of carbon coated material per microliter, wherein thecarbon coated material is preferably magnetic or silica nanoparticulatefor this embodiment. The suspension for storage contains between 20% and80% alcohols. The single-strand nucleic acid, while on the surface ofthe carbon-based material such as graphene, cannot be cleaved by enzymessuch as RNase and will not spontaneously degrade.

Aspects of the disclosed implementations also include a method forfabricating the carbon coated magnetic beads that can be employed in themethod and process disclosed herein. One such fabrication method isdiscussed in US Published Application Number 2015-0170807 to Hagedorn,K. and Malizia, H, the specification of which is incorporated byreference herein. The referenced patent discloses methods for preparingmagnetic beads. The magnetic beads prepared in carbon containing organicsolvents such as xylenes or toluene may create a carbon coating on thesurface of the magnetic beads suitable for use in the disclosed methods.

Example I

To further illustrate the nucleic acid separation method describedherein, the process was used to separate RNA from samples of 1 millionMCF7 breast cancer cells with various process parameters and the RNAseparated was quantified with gel electrophoresis in FIG. 7. Samples of1 million MCF7 breast cancer cells were introduced into respectivesterile glass containers at standard temperature and pressure. 125 μl ofguanidinium thiocyanate solutions were added to each respective samplewhere the concentrations of guanidinium thiocyanate were 2M at reference702, 3M at reference 703, 4M at reference 704, 5M at reference 705, and6M at reference 706. The guanidinium thiocyanate solution also contained1% Triton X-100, 10 mM EDTA, and 20 mM Tris, and the pH was adjusted to6. Graphene coated magnetic beads as shown in FIG. 5 are then added tothe respective samples. A solution of 70% ethanol is added to eachsample to a constant 56% of the solution by volume, making theconcentration of the guanidinium thiocyanate 0.4M at reference 702, 0.6Mat reference 703, 0.8M at reference 704, 1M at reference 705, and 1.2Mat reference 706 respectively. The graphene coated magnetic beads are 20nm to 100,000 nm in size and have a carbon layer 1 angstrom to 50 nmthick. The carbon layers can be visualized in FIG. 5 as rings around themagnetic metal core. The magnetic metal core may be any material whereFe, Co, or Ni individually or combined compose more than 30% of materialby atomic percent. The supernatant was separated from the carbon-coatedmagnetic beads complexed with nucleic acids and washed three times witha solution of 70% ethanol. 100 μl of purified water was then added andthe single stranded nucleic acids were released into this water.

Portions of 10 μl of each respective sample was removed and subjected toelectrophoresis. An agarose gel was cast and the samples were mixed withan ethenium bromide loading dye. The gel was run at 90V for 1 h.

This non-limiting example exemplifies how the process is industriallyapplicable by demonstrating RNA isolation from cells without DNAcontamination. This non-limiting example also exemplifies the unexpectedresult that single stranded nucleic acids are extracted withoutcontamination from double stranded nucleic acids when the concentrationof chaotropic salts is between 0.1M and 1M.

Example II

To further illustrate the nucleic acid separation method describedherein, the process was used to separate RNA from samples of 1 millionMCF7 breast cancer cells with various process parameters and the RNAseparated was quantified with gel electrophoresis in FIG. 8. Samples of1 million MCF7 breast cancer cells were introduced into respectivesterile glass containers at standard temperature and pressure. 125 μl of2M guanidinium thiocyanate solutions were added to each respectivesample. A solution of 70% ethanol is added such that the concentrationof ethanol is 0% at reference numeral 801, 31% by volume at referencenumeral 802, 43% by volume at reference numeral 803, 49% by volume atreference numeral 804, and 56% by volume at reference numeral 805. It isobserved that when 6M guanidinium thiocyanate solution is provided withcarbon coated magnetic beads but without ethanol as previouslydescribed, single stranded nucleic acids are separated from the mixturebut with poorer yield. The addition of ethanol has the unexpected resultof enhancing recovery yield.

Portions of 10 μl of each respective sample was removed and subjected toelectrophoresis. An agarose gel was cast and the samples were mixed withan ethidium bromide loading dye. The gel was run at 90V for 1 h.

This non-limiting example exemplifies how the process is industriallyapplicable by demonstrating RNA isolation from cells without DNAcontamination. This non-limiting example also exemplifies the unexpectedresult that single stranded nucleic acids are extracted withoutcontamination from double stranded nucleic acids when the concentrationof alcohols is between 20% and 80% of the solution by volume.

Example III

FIG. 9 compares RNA extraction from 1 million MCF7 breast cancer cellsusing process outlined in Example I in the first lane 901 to the ThermoFisher MirVana Total RNA Extraction Kit using the procedure described inthat kit in the second lane at reference numeral 902. The Thermo FisherMirVana Total Extraction Kit is a state-of-the-art kit from one of thelargest life science suppliers in the world. The most noticeabledifference is the absence of DNA contamination. DNA contamination is aproblem for RNA analysis because PCR amplification cannot be performedwith DNA contamination. Heretofore, the DNA contamination had to beremoved with DNase, an enzyme which destroys DNA. This is both expensiveand slow. The second noticeable difference is improved yield of smallRNA, which is important for applications which analyze short RNA such asMicro RNA for diagnostic and research applications.

While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiment,it is to be understood that the invention is not to be limited to thedisclosed embodiments but, on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims, which scope is to be accorded the broadestinterpretation so as to encompass all such modifications and equivalentstructures as is permitted under the law.

1. A method for separating single stranded nucleic acids from associatedbiological material, the method comprising: creating a complex of acarbon coated material and single stranded nucleic acids from a mixturecontaining single stranded nucleic acids, double stranded nucleic acids,the carbon coated material, at least one chaotropic salt and at leastone alcohol having between one and four carbon atoms, wherein thematerial is a chaotropic salt present at a concentration from 0.01M to3M and wherein the alcohols are present as 5 vol % to 99 vol %; andafter the complex of carbon coated material and single stranded nucleicacids has been created, removing the carbon coated material complexedwith single-stranded nucleic acid from the mixture.
 2. The method ofclaim 1 wherein the carbon coating present on the carbon coated materialis at least one of graphene, pyrolytic carbon or a mixture of grapheneand pyrolytic carbon.
 3. The method of claim 1, wherein chaotropic saltis a guanidinium salt composed of a guanidinium cation and an anionselected from the group consisting of chloride, bromide, fluoride,acetate, sulfate, nitrate, carbonate, thiocyanate and mixtures thereon.4. The method of claim 1, wherein the chaotropic salt is present at aconcentration between 0.01 M and 0.6M.
 5. The method of claim 1, whereinthe alcohol is present in an amount between 10 vol % and 85 vol %.
 6. Amethod for isolating nucleic acids having a defined sequence from anaqueous solution, the method comprising: contacting a compositioncontaining single stranded nucleic acid and double stranded nucleic acidwith at least one nucleic acid probe, the nucleic acid probecomplimentary with amino acids present on at least a portion of thenucleic acid in the aqueous solution, the contacting step occurring foran interval sufficient for the at least one nucleic acid probe to duplexwith amino acid sequences present on at least a portion of the nucleicacid present in the solution to produce an admixture composed ofduplexed nucleic acid material and non-duplexed nucleic acid material;to the resulting admixture containing duplexed nucleic acid material,adding carbon-coated material, at least one chaotropic salt andshort-chain alcohol, the short-chain alcohol present in an amountbetween 2 vol % and 99 vol %; allowing contact for an intervalsufficient to permit association between the carbon coated material andat least a portion of the single stranded nucleic acids present in theadmixture; separating carbon-coated material complexed with the singlestranded nucleic acids from liquid supernatant, the liquid supernatantcontaining complexed double stranded nucleic acid.
 7. The method ofclaim 6, wherein the carbon coated material is configured as beads, thebeads each having a core and an outer surface wherein at least the coreis magnetic and contains at least 30% by weight of a metal selected fromthe group consisting of Ni, Fe, Co, or mixtures thereof.
 8. The methodof claim 6, wherein the chaotropic salt is added in an amount sufficientto provide a concentration between 0.01M and 3M.
 9. The method of claim8, wherein chaotropic salt is selected from the group consisting of aguanidinium salts, urea, thiourea, lithium acetate, lithium perchlorate,magnesium chloride and mixtures thereof.
 10. The method of claim 9,wherein chaotropic salt is a guanidinium salt composed of a guanidiniumcation and an anion selected from the group consisting of chloride,bromide, fluoride, acetate, sulfate, nitrate, carbonate, thiocyanate andmixtures thereof.
 11. The method of claim 10, wherein the concentrationof the chaotropic salt is between 0.01 and 8M.
 12. The method of claim6, wherein the short chain alcohol is selected from the group consistingof methanol, ethanol, propanol, isopronaol, phenol, and mixturesthereof.
 13. The method of claim 6, wherein the removing step includessubjecting the resulting complex of carbon-coated magnetic beads andnucleic acid to at least one of a magnetic field, a centrifugal force,precipitation, competitive binding, or mixtures thereof.
 14. The methodof claim 6, wherein the concentration of alcohols is between 10% and95%.
 15. The method of claim 6, wherein the carbon coated materialcomprises a substrate and an outer carbon coating, the carbon coatinghaving a thickness between 1 angstrom and 50 nm.
 16. The method of claim6, further comprising: after removing the carbon coated magnetic beadsfrom the supernatant, releasing single-stranded nucleic acid fromattachment to the carbon coated material, wherein the releasing stepoccurs with addition of water or suitable buffer solution into contactwith the removed carbon coated material.
 17. The method of claim 16wherein the removal step includes at least one of the following:centrifugation, precipitation, competitive binding, subjecting thecomposition to a magnetic field.
 18. The method of claim 6, wherein thecarbon-coated material is configured as individual beads, each beadhaving an interior core and an outer surface, the beads composed of oneof the following: a magnetic metal containing at 30% by weight of ametal selected from the group consisting of Ni, Fe, Co, or mixturesthereof, silica or polymeric substrates, and wherein the carbon coatingis present as a carbon layer on at least a portion of the outer surfaceof the bead, the carbon layer comprising at least one of graphene,pyrolytic carbon or a mixture of graphene and pyrolytic carbon, andwherein the multivalent cations are alkali earth metals or alkali earthmetal salts or mixtures thereof.
 19. The method of claim 6, wherein thecomplementary nucleic acid probe is a chain of nucleic acids selectedsuch that the chain of nucleic acids contains complimentary bases withcorresponding positions to the single stranded nucleic acid that is tobe isolated or purified.
 20. The method of claim 6, wherein thecomplementary nucleic acid probe may be DNA or a synthetic nucleic acidand wherein the method further comprises the step of quantifying thesequence for study by performing PCR on the supernatant which remainsafter removal of carbon coated magnetic material and associated singlestranded nucleic acids.